The response of eukaryotes to DNA damaging agents, such as ionizing radiation, is determined by the effectiveness of a variety of DNA repair systems. Defective repair, in the form of a diminished capacity for accurate repair or through inaccurate repair processes, can lead to mutations involving genes responsible for the control of cell proliferation. Understanding the nature of these repair processes is central to the elucidation of the mechanisms through which subsequent adverse health effects such as mutations, fetal malformations, and cancers are expressed. Ionizing radiation induces various types of DNA damage in mammalian cells including DNA single-strand breaks (SSB), DNA double strand breaks (DSB), DNA-protein crosslinks, DNA-DNA interstrand crosslinks, and altered DNA bases. Although- it is obvious that human cells can repair many of these lesions, there is little detailed knowledge of the nature of the genes and the encoded enzymes that control these repair processes. ln order to understand how DNA repair modulates radiation sensitivity, oncogene expression, and recombination-related genetic diseases, radiation-specific DNA repair genes have to be isolated and characterized. During the current funding period of this project, a comprehensive strategy and all the basic grounds for the positional cloning of a human DSB repair gene XRCC5 have been established in our laboratory. Thus, the primary objective of this renewal application is to continue our efforts on the isolation and characterization of human XRCC5 gene. The specific aims are: 1) Regional localization of human XRCC5 gene in chromosome 2 (at the megabases level); 2) Construction of a repair-proficient X-ray hybrid panel, each hybrid containing a fragment of human chromosome 2 (1-3 Mb) where the XRCC5 has been assigned ;3) Position cloning of candidate XRCC5 recombinant clones and functional analysis of these clones; 4) Molecular characterization of the cloned XRCC5 gene. We believe that this step-by- step approach utilizing advanced molecular genetic technologies will substantially facilitate the isolation of the first human radiation repair gene(s) responsible for the rejoining of ionizing radiation-induced DNA double-strand breaks.